Method for sex determination of mammalian offspring

ABSTRACT

A method for increasing the percentage of mammalian offspring of either sex which comprises contacting a semen sample with an antibody specific for the spermatozoa determinative of one sex and separating said spermatozoa from spermatozoa determinative of the other sex, said antibody being bound to a non-porous magnetic bead support having a diameter of 0.1 to 2 microns.

This application is a divisional application of co-pending applicationSer. No. 09/299,181, filed on Apr. 23, 1999, now issued as U.S. Pat. No.6,153,373, which is a continuation of co-pending application Ser. No.08/886,203, filed on Jul. 1, 1997, and now abandoned the entire contentsof which are hereby incorporated by reference.

BACKGROUND AND FIELD OF THE INVENTION

The present invention is directed to a method for increasing thepercentage of mammalian offspring of either sex by contacting a spermsample with an antibody specific for one sex, the antibody being boundto a magnetic bead of a diameter which permits separation of spermatozoahaving sufficient motility to permit successful insemination andfertility.

Farmers and other animal husbandry persons have long recognized thedesirability of enhancing the probability of offspring of a selectedsex. Methods have been proposed in the past for increasing thepercentage of X-sperm cells or Y-sperm cells to thereby achieve agreater chance of achieving male or female offspring, respectively.Examples of prior research are reviewed, for example, in Garner, D. L.et al., “An Overview of Separation of X- and Y-Spermatozoa,” Proceedingsof the Tenth Technical Conference on Artificial Insemination andReproduction (National Association of Animal Breeders), pp. 87-92 (1984)and Pinkel, D. et al., “Flow Cytometric Determination of the Proportionsof X- and Y-Chromosome Bearing Sperm In Samples of Purportedly SeparatedBull Sperm,” J. Animal Scien., 60, pp. 1303-1307 (1985).

Previous methods have included, for example, methods based upon densitysedimentation (see, for example, Brandriff, B. F. et al., “SexChromosome Ratios Determined by Karyotypic Analysis in Albumin-IsolatedHuman Sperm,” Fertil. Steril., 46, pp. 678-685 (1986)).

U.S. Pat. No. 3,687,806 to Van Den Bovenkamp discloses an immunologicalmethod for controlling the sex of mammalian offspring by use ofantibodies which react with either the X- or Y-chromosomes and utilizingan agglutination step to separate bound antibodies from unaffectedantibodies.

U.S. Pat. No. 4,191,749 to Bryant discloses a method for increasing thepercentage of mammalian offspring of either sex by use of amale-specific antibody coupled to a solid-phase immunoabsorbant materialto selectively bind male-determining spermatozoa, while thefemale-determining spermatozoa remain unbound in a supernatant.

U.S. Pat. No. 5,021,244 to Spaulding discloses a method for sortingliving cells based upon DNA content, particularly sperm populations toproduce subpopulations enriched in X- or Y-sperm by means ofsex-associated membrane proteins and antibodies specific for suchproteins.

However, these methods often result in insufficient separation of X- andY-sperm and often damage the sperm, thereby reducing its motility andfertility success rate.

SUMMARY OF INVENTION

It is, therefore, an object of the present invention to provide a methodfor increasing the probability that mammalian offspring will be of aspecific desired sex.

It is a further object of the invention to provide a method for theseparation of X- and Y-determinative spermatozoa without compromisingthe motility or fertilization rate of the separated spermatozoa.

It is another object of the present invention to provide a method forartificial insemination which permits insemination with a sperm sampleenriched in X- or Y-determinative sperm.

It is still a further object of the invention to provide a method forseparating X- and Y-determinative sperm by means of antibodies specificfor the selected sperm, bound to magnetic beads, to permit highlyspecific separation, so as to provide a sperm sample enriched in oneselected spermatozoa type and substantially free of the otherspermatozoa type.

These and further objects of the present invention are achieved by amethod which contacts a sperm sample with antibodies specific to aselected spermatozoa type, the antibodies being bound to beads,preferably magnetic beads having a diameter of from 0.1 to 2 microns,and subsequently removing the beads whereby the remaining supernatantmay be collected and contains spermatozoa of only X- or Y-determinativetype.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for separation of X- and Y-bearing spermwhich are competent to fertilize using standard Al techniques. As notedabove, prior methods of separation often compromise the motility andfertilization ability of the sperm, so that fertilization utilizing suchseparated sperm requires complicated techniques such as IVF. The methodof the invention can be utilized for separating X- and Y-sperm from avariety of mammalian species, including various livestock, such ascattle and sheep, as well as dogs, cats, horses, swine, and otherspecies. The process is also applicable to humans.

By means of the present invention, a sperm sample containing both X- andY-sperm can be separated to produce an X- or Y-enriched spermsubpopulation which is substantially pure with respect to the desiredspermatozoa and substantially free of the other spermatozoa-type. By“substantially free,” we mean that use of a sample enriched, forexample, with X-sperm, when utilized for artificial insemination, hasonly a small chance of producing male offspring, because the spermsample has less than 20%, preferably less than 10%, of Y-sperm.Separation of the X- or Y-spermatozoa is accomplished by use ofantibodies which bind to X- or Y-specific proteins from sperm cells.These antibodies can be of any type of antibody (including IgG and IgM)and can be either polyclonal antibodies or monoclonal antibodies. Ifpolyclonal antibodies are to be used, then such antibodies can beprepared according to per se known procedures. For example, proceduressuch as those described in Hurn, B. A. et al., (1980), Meth. inEnzymology, Ed. Van Vanakis, H. and Langone, J., pp. 104-142, can beused.

If desired, monoclonal antibodies can be utilized and prepared accordingto methods which are per se known in the art, such as those originallyauthored by Milstein and Kohler, published in Nature (1975), 256, pp.495-497. This basic procedure involves injecting an animal, usually amouse, with an immunogenic substance. After suitable time for antibodyproduction to the immunogen, the mouse is sacrificed. Cells are removedfrom the spleen and fused with myeloma cells. Hybridoma cells resultingfrom this fusion are able to reproduce in vitro, and each express agenetic information for one specific antibody. The antibodies producedfrom one hybridoma fusion thus will only recognize a single antigenicdeterminative of the immunogen.

Cells cultured from individual hybridoma cells can then be screened forproduction of antibodies to the target antigenic determinant. Thosehybridomas positive for the target antigen can be further screened toidentify those having the desired level of affinity. Monoclonalantibodies displaying all of these characteristics can then be screenedusing actual assay conditions to determine if the assay condition altersthe antibody binding characteristics or affinity, and to screen outthose with cross-reactivity to possible contaminating antigens.

Preferred antibodies are those which are specific for and bind toY-sperm, such as antibodies which bind to the H-Y antigen. Suchantibodies can be prepared, for example, by the procedure described inU.S. Pat. No. 4,680,258 to Hammerling et al.

As noted above, the antibodies specific for either X- or Y-spermatozoaare immobilized on beads. These beads can be plastic beads or magneticbeads. Useful plastic beads are SEPHAROSE™ 6MB, or other beads which arelarge enough to settle out in a batch purification process. When plasticbeads are utilized, the beads having antibody bound thereto are mixed ina sperm sample and allowed to settle to the bottom of the container.This step can be repeated, if desired, to increase the completeness ofseparation of sperm according to sex chromosome.

If magnetic beads are used, the beads are microspheres of magneticparticles representing an immobilizing matrix. It has been found,according to the present invention, that magnetic beads having adiameter of from 0.1 to 2 microns in diameter are specifically usefulfor separating the desired species of spermatozoa without compromisingthe motility and fertilization ability of the spermatozoa. Particularlyuseful magnetic beads are described, for example, in U.S. Pat. No.5,071,076; U.S. Pat. No. 5,108,933; U.S. Pat. No. 4,795,698; and PCTPatent Publication No. WO91/09678. According to the procedures describedin these patents, beads can be prepared having especially a diameter of0.1 to 0.5 microns.

The antibodies are bound to the beads by means of procedures which areper se known in the art. In general, a linking compound is attached tothe magnetic beads during manufacture of the beads. On to the beads, alinking compound such as an antibody (such as an IgG antibody which isdirected against mouse IgM) is bound by mixing beads at about 1 mgiron/ml with purified antibody at 1 mg/ml protein. After the antibody isbound to the beads, the beads are washed so only attached antibodyremains. Additional procedures known to those skilled in the art aredescribed, for example, in U.S. Pat. No. 4,018,886; U.S. Pat. No.3,970,518; U.S. Pat. No. 4,855,045; and U.S. Pat. No. 4,230,685. ProteinA is a preferred linking compound which greatly increases theeffectiveness of capture by the attached antibodies. (Forsgren et al.(1977) J. Immunol. 99: 19). Protein A attaches to the Fc portion of IgGsubclass antibodies, thus extending and presenting the Fab portion ofthese antibodies. The resulting correct orientation of the antibodiesand extension away from the particles leads to a very effectiveinteraction between the bound antibodies and their target.

The method of attachment of Protein A to magnetic particles may proceedby any of several processes available through known scientificliterature. In one such procedure, magnetic iron oxide particles ofapproximately one micrometer diameter are chemically derivatized by areaction, first with 3-aminopropyltri-ethoxysilane, then withglutaraldehyde. The derivatized magnetic particles are then mixed withProtein A resulting in a magnetic particle to which Protein A iscovalently attached. The antibodies are then added to the Protein Amagnetic particles and after a short incubation, the Protein A-antibodycomplexes form. (Weetall, H. H. (1976) Meth. In Enzymol. 44:134-48).

The following is a specific example of the method according to theinvention.

EXAMPLE 1

About 10⁸ to 10¹⁰ spermatozoa in amount 1-10 ml of volume are washedwith buffered culture medium (Dulbeco's Modified Eagles Medium) bycentrifuging at 1,500 rpm for 10 minutes. The sperm are then resuspendedin 4 ml buffered culture medium after gently decanting the supernatant.The sperm suspension is then divided into two vials, and into each vialis added 500 iL of magnetic beads having bound thereto antibodies forY-bearing sperm. The vials are capped and then rotated at 3 rpm, endover end, on a rotator at room temperature for 30 minutes.

After rotation, the caps are loosened and placed in a magnetic capturerack, such as that described in U.S. Pat. No. 5,571,481. After 15minutes of exposure to the magnetic field, the supematant is removedwhich is rich in X-bearing sperm and substantially free of Y-bearingsperm. The removed supernatant is then counted for sperm, and theconcentration is adjusted as necessary for insemination. That sample isthen loaded into a straw or straws for the insemination procedure.

If desired, more complete separation can be obtained by repeating, in aserial manner, the magnetic capture procedure on the supernatant.

Each of the publications/patents referred to above is herebyincorporated by reference.

The invention being thus described, it will be clear that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A method for separating spermatozoa determinativeof one sex from spermatozoa determinative of the other sex comprisingcontacting a semen sample with antibody specific for a spermatozoadeterminative of one sex, and magnetic beads having a diameter of 0.1 to2 microns, and separating spermatozoa bound to said antibody and linkedto said beads from said sample.
 2. The method according to claim 1,wherein said antibody is linked to said beads through a linkingcompound.
 3. The method according to claim 2, wherein said linkingcompound is an antibody.
 4. The method according to claim 3, whereinsaid linking antibody is IgG.
 5. The method according to claim 2,wherein said linking compound is protein A.
 6. The method according toclaim 5, wherein said semen sample is contacted with said beads, thesample is exposed to a magnetic field, and the remaining supernatant isremoved to provide a supernatant rich in X-bearing sperm.
 7. The methodaccording to claim 6, wherein said supernatant rich in X-bearing spermis subjected to said method at least one additional time.
 8. The methodaccording to claim 1, wherein said antibody specific for saidspermatozoa determinative of one sex is IgG.
 9. The method according toany one of claims 1-5 or 8, wherein said antibody specific for saidspermatozoa determinative of one sex is specific for Y-bearing sperm.10. The method according to claim 9, wherein said antibody specific forY-bearing sperm is a polyclonal antibody.
 11. The method according toclaim 9, wherein said antibody specific for Y-bearing sperm is specificfor an H-Y antigen.
 12. The method according to claim 9, wherein saidbeads have a diameter of 0.1 to 0.5 microns.
 13. The method according toclaim 12, wherein said antibody specific for Y-bearing sperm is amonoclonal antibody.
 14. The method according to any one of claims 1-5or 8, wherein said antibody specific for said spermatozoa determinativeof one sex is specific for X-bearing sperm.
 15. The method according toclaim 14, wherein said beads have a diameter of 0.1 to 0.5 microns. 16.The method according to claim 15, wherein said antibody specific forX-bearing sperm is a monoclonal antibody.
 17. The method according toclaim 14, wherein said antibody specific for X-bearing sperm is apolycolonal antibody.
 18. The method according to claim 1, wherein saidsemen sample is contacted with said beads, the sample is exposed to amagnetic field, and the remaining supernatant is removed to provide asupernatant rich in Y-bearing sperm.